Ras proteins are key molecular switches that transmit signals to control a variety of cellular processes including proliferation, differentiation, and survival. Precise regulation of the intensity and duration of Ras-mediated signaling is critical fo normal physiological responses as well as for disease prevention. The goal of this proposal is to uncover novel and fundamentally important mechanisms for Ras regulation, using both the genetically tractable model organism yeast and the complimentary system of human cell biology. The main hypothesis is that Ras signaling is regulated via dynamic ubiquitination and deubiquitination of RasGAP. The hypothesis is based on recent observations that RasGAP (Ira2) is ubiquitinated and disrupting its interacting deubiquitinase (Ubp3) markedly enhances Ras activation. Three specific aims are proposed: Aim 1: How does Ubp3 inhibit Ras signaling? Disrupting Ubp3 leads to hyperactivation of Ras; Ubp3 interacts with Ira2, a negative regulator of Ras that undergoes ubiquitination and degradation. Our hypothesis is that Ubp3 inhibits Ras signaling via deubiquitinating Ira2 and protecting it from degradation. To test this, we will characterize the interaction between Ubp3 and Ira2, and examine whether Ubp3 deubiquitinates Ira2 and affects its abundance, stability, or subcellular localization. To identify other targets o Ubp3, we will systematically identify components in the pathway that are required for Ubp3 to exert its effect on signaling. Any component identified will be further characterized. Aim 2: What are the ubiquitinating enzymes that targets RasGAP? Despite the key role of RasGAP ubiquitination in regulating Ras, the responsible ubiquitin ligating enzymes remain elusive. To identify the enzymes that ubiquitinate Ira2, initially we will consider Gpb1, a protein known to play a role in Ira2 ubiquitination. To identify additional factors required for Ira2 ubiquitinationand normal signaling, we will systematically examine yeast strains lacking known or suspected ubiquitinating enzymes for loss of Ira2 ubiquitination and for an alteration of Ras-mediated signaling. Any proteins identified will be further analyzed using in vitro assay for their ability o ubiquitinate Ira2. Aim 3: Is human Ras signaling regulated by deubiquitination? Ubp3 has a human homolog Usp10, a protein implicated as a potential tumor suppressor. To determine if the mechanisms elucidated in yeast is conserved in humans and to provide a new perspective for considering the role of Usp10 in tumorigenesis, we will examine if Usp10 regulates Ras signaling in humans. Specifically, we will test whether silencing Usp10 expression affects Ras signaling in human cells, and whether it does so via deubiquitinating and protecting RasGAP neurofibromin (Ira2 homolog in human) from degradation.